This article, originally published Oct.7, has been updated with comments from an author of the study.

A primary breast cancer acquired several new mutations in its protein-coding regions as it progressed toward metastasis, according to a new report by Canadian researchers, who argue that studying these mutations might help scientists understand why cancers become treatment resistant.

The research team, of the BC Cancer Agency in Vancouver, sequenced the genome and transcriptome of a metastatic breast cancer at high depth, using the Illumina Genome Analyzer platform, and analyzed the data for somatic coding mutations.

The scientists then determined how many of these mutations they could already detect in the primary tumor of the same patient, which was removed nine years earlier. Only a fraction of them could be found at all, and even fewer were present at a high frequency.

In addition, by analyzing not only genome but also transcriptome data, they uncovered two new RNA-editing events that change the amino acid sequence of two proteins.

The study “offered the opportunity, for the first time as far as we are aware, to look at the evolution of mutational burden across a very long period of time within the same patient,” said Marco Marra, director of the BC Cancer Agency Genome Sciences Centre in Vancouver, and one of the authors of the report, which appeared online in Nature last week.

“Our results show the importance of sequencing samples of tumor cell populations early as well as late in the evolution of tumors, and of estimating allele frequency in tumor genomes,” the authors noted.

Being able to study how a metastatic cancer’s genome differs from that of a primary tumor “is important because it gets at the heart of how treatment shapes the genetic
constitution of a malignancy,” Marra said. “We need to know, with great precision, what the changes are that allow tumors to evade treatment.”

The project, which was completed about six months ago, according to Marra, was just the beginning of a large-scale effort to study treatment-resistant cancer. “We are going to continue with using large-scale high-throughput approaches, such as DNA sequencing, to get at the mutational spectrum of treatment-resistant [cancers],” he said, including breast cancer, hematologic malignancies, childhood cancers, and lung cancer.

For their study, the Canadian researchers chose lobular breast cancer, an estrogen-receptor positive subtype that makes up about 15 percent of all breast cancers.

One of the reasons for focusing on breast cancer as the first example is that the Vancouver researchers plan to study more breast cancer samples as part of the Molecular Taxonomy of Breast Cancer International Consortium, or METABRIC, project, a collaboration between five hospitals and research centers in the UK and Canada, Marra said.

According to the BC Cancer Foundation, the Vancouver team is now sequencing the genomes of several hundred so-called triple negative breast tumors as part of an effort to build “a comprehensive genomic map of breast cancer” from 2,000 samples.

For their published study, the researchers initially sequenced DNA from a metastatic lobular breast cancer sample, generating approximately 2.9 billion paired-end reads with a mean read length of 48 base pairs, or 141 gigabases of sequence data, on the Illumina Genome Analyzer. About 121 gigabases aligned to the human reference genome, equivalent to about 43-fold coverage.